Systematic Mutagenesis

Site-saturation mutagenesis is known to systematically replace wild type amino acid coding sequences with sequences encoding all 19 non–wild type amino acids at the position(s) you specify. Also known as sequential permutation, site-saturation mutagenesis in combination with a powerful screening assay is the most systematic mutation strategy to identify amino acid substitutions that fulfill your protein engineering goals.

Once you’ve identified variants with beneficial substitutions, the GeneArt™ Combinatorial Library Service can be used to create material for more targeted directed-evolution experiments to further optimize your protein.

Case study: Functional Mapping of RNA Polymerase (Customer Example)

To determine the impact of structural changes on RNA polymerase enzyme activity Tan et al.characterized the functional properties of mjA´ subunit mutants. A subset of mutants were produced by GeneArt site-saturation mutagenesis services.

Activity testing of the resulting single-point mutants demonstrated a wide spectrum of different phenotypes (see graphic below), including some hyperactive variants having higher activity than wild type. These were subjected to further investigations.

More details can be found in the original article, which can be accessed here.

Options for Site-Saturation Mutagenesis

We offer several options for sequential permutation to fit a wide array of research needs. You can choose to create variants at one or more codons, and receive constructs individually or pooled. When evaluating most of the possible 19 non–wild type amino acids is sufficient, we offer the GeneArt™ Site-Saturation Mutagenesis Average 16 and GeneArt™ Site-Saturation Mutagenesis Minimum 16 Services that provide individual clones encoding an average of 16 or a minimum of 16 substitute amino acids at each position, respectively.

Variant constructs are subcloned into the vector of your choice

GeneArt™ Site-Saturation Mutagenesis Pool of One Position

A mixture of all 19 non–wild type variants at one codon position

Supplied as a pooled glycerol stock

GeneArt™ Site-Saturation Mutagenesis Pool of All Positions

A mixture of all 19 non–wild type variants at more than one codon position